This application claims priority from Swedish Patent Application No 9601245-5, which was filed Mar. 29, 1996, and is incorporated herein by reference.
The present invention relates to functionally active modified superantigens which are wild-type superantigens (SA I) in which one or more amino acid residues have been substituted while maintaining superantigen function. In case one or more of the substituting residues (or a conserved amino acid residue thereof) occur in the corresponding positions in another wild-type superantigen (SA II), the modified superantigen is called a chimera. Chimeric superantigens thus will contain part sequences/regions deriving from at least two different wild-type superantigens.
By the term xe2x80x9ccorrespondingxe2x80x9d is meant that residues, part sequences and regions replacing each other have functionally the same position in superantigens I and II so that substitution will lead to a chimeric form that is able to function as a superantigen.
The terminology grafted/grafting/graft is used in connection with parts of the full sequence of superantigen II that have replaced corresponding parts of superantigen I, even if only one single amino acid has been replaced.
Modified/chimeric superantigens also encompass functional superantigens modified in other ways, for instance conjugated to a target-seeking moiety, including also fused forms when the moiety is a polypeptide/protein. See below.
Superantigens
According to the very first definition (around 1988-1993), superantigens are bacterial or viral proteins capable of binding to MHC class II antigens without prior intracellular processing and activate T cells by binding to the xcex2-chain variable region (V xcex2) of the T cell receptor (TCR). The binding leads to a Vxcex2 family restricted activation of a relatively large proportion/subset of T cells and lysis of MHC Class II expressing cells (superantigen dependent cell cytolysis=SDCC).
Well known wild-type superantigens according to the definition above are the staphylococcal enterotoxins (SEA, SEB, SEC1, SEC2, SED, SEE and SEH). Further examples are Toxic Shock Syndrome Toxin 1 (TSST-1, also of staphylococcal origin), Exfoliating Toxins (EXft), Streptococcal Pyrogenic Exotoxin A, B and C (SPE A, B and C), Mouse Mammary Tumor Virus proteins (MMTV), Streptococcal M proteins, Clostridial Perfringens Enterotoxin (CPET), mycoplasma arthritis superantigens etc. For a review of superantigens and their properties see Kotzin et al 1993.
During the early nineties it was discovered that activation and subsequent cell lysis could occur in a MHC class II independent manner in case the superantigen was conjugated with a target-seeking moiety capable of binding to a cell surface structure (Dohlsten et al WO9201470 and Abrahmsxc3xa9n et al WO9601650). Upon incubation of target cells (carrying the target structure for the target-seeking moiety) and effector cells (T cells) with the conjugates, the target cells become lysed (superantigen antibody dependent cell cytolysis=SADCC) without any requirement for class II expression. Accordingly the superantigen concept of today and used in the context of the present invention, if not otherwise specified, encompasses any compound (preferably of polypeptide structure) that is capable of binding to a cell surface structure (target structure) and to one or more polymorphic TCR chain, in particular the Vxcex2 chain, thereby activating a subset of T cells expressing the specific TCR chain involved in the binding. The T cells then become cytotoxic for cells carrying the surface structure (target structure, target cells). Normally the activated subset of T cells constitutes about 1-20% of the total amount of T cells of an individual.
Background Artxe2x80x94Structural and Functional Studies Utilizing Mutated and Chimeric Superantigens
Chimeric superantigens including point mutated forms have previously been described (Kappler et al WO 9314364, Kappler et al 1992; Grossman et al 1991; Hufnagle et al 1991; Hartwig et al 1993; Fraser et al 1993; Mollick et al 1993; Erwin et al 1992; and Hudson et al 1993). Mollick et al and Hudson et al show from studies of chimeras that the Vxcex2 specificity of SEA and SEE resides in certain amino acid sequences present in the carboxy terminal region (i.e. amino acid residues 200, 206 and 207). In addition to the Vxcex2 specificity, mainly depending on this region, Mollik et al also were able to show that for complete reconstitution of SEE like activity of SEA containing SEE grafts towards Vxcex28, a fragment containing the N-terminal 70 amino acid residues from SEE was needed. This fragment contains parts of the SEE-like MHC class II xcex1 chain binding site and chimeric SEA/SEE molecules containing this part from SEE, inhibited binding of SEA to MHC class II DR1 in a SEE-like manner.
Recently SEE-SEA chimers involving an exchange of regions involved in binding to TCRVxcex2 have been described (Lamphaer et al., J. Immunol. 156 (Mar. 15, 1996) 2178-2185). A SEE superantigen Fab antibody fusion protein in which the SEE domains involved in the interaction with T cells have been replaced with the corresponding non-homologous SEA domains has been discussed at ABRF""96: Biomolecular Techniques, Holiday Inn Golden Gateway, San Francisco, Calif. Mar. 30-Apr. 2, 1996 (Bjxc3x6rk et al., M45).
Background Artxe2x80x94Therapeutic Use of Superantigens
Non-conjugated superantigens have been suggested for therapy with curative effect presumably being accomplished through a general activation of the immune system (Kalland et al WO9104053; Terman et al WO9110680 and WO9324136; Newall et al 1991).
It has also been suggested to use modified superantigens conjugated to target-seeking moieties (Dohlsten et al WO9201470; Abrahmsxc3xa9n et al WO9601650, both hereby being incorporated by reference). This enabled a broader therapeutic use of T cell activation through Vxcex2. The conjugates studied so far have had a diminished class II affinity, which in turn has lead to a decrease of the severe systemic toxicity normally associated with the wild-type superantigens.
Terman et al (WO9110680; WO9324136) in side-sentences suggested cancer therapy with modified superantigens and superantigen fragments.
Kappler et al (WO9314634) have suggested to use non-conjugated to superantigens mutated to have lost their Vxcex2-binding ability (in the context of vaccines). Abrahmsxc3xa9n et al (WO9601650) have suggested cancer therapy with conjugated superantigens having a modified, preferably decreased, ability to bind to Class II antigens. The modifications encompassed single mutations as well as construction of chimeras between different superantigens.
The Problems that Have Been the Objective to Solve with the Present Invention
The sera of human populations normally contain high titers of antibodies against superantigens. For the staphylococcal superantigens, for instance, the relative titers are TSST-1 greater than SEB greater than SEC1 greater than SE3 greater than SEC2 greater than SEA greater than SED greater than SEE. These relative titers indicate immunogenicity problems and problems with neutralizing antibodies in case SEs are administered parenterally. Based solely on these problems, SEE should be the preferred staphylococcal superantigen. In the context of work with fusion proteins, however, we have found that the ability for T cell MHC class II independent cytotoxicity, superantigen-antibody dependent cell cytotoxicity (SADCC), of SEE conjugates is poor. The anti-SE titers also indicate that there might be advantages in modifying a xe2x80x9chigh titerxe2x80x9d superantigen to be more like a xe2x80x9clow titerxe2x80x9d superantigen.
The Objectives of the Present Invention
A first objective is to improve the previously known superantigens with respect to lowering their immunogenicity and reaction with neutralizing antibodies.
A second objective is to provide superantigens with less side effects when used as a drug.
A third objective is to provide improved superantigens that can be used as the active principle in the treatment of mammals suffering from cancers, autoimmune diseases, parasitic infestations, viral infections or other diseases associated with cells that on their surface express MHC class II antigens and/or structures that are specific for respective disease and bind to a target-seeking moiety incorporated into the superantigen.
The Discovery that has Resulted in the Invention
A sequence homology analyzis of SEA and SEE (FIG. 2) reveals that the non-identical amino acid residues are concentrated to as eight distinct regions. These regions are identified by A, B, C, D, E, F, G, and H as depicted in FIG. 2. For SEA, and SEE the sequences in these regions are identified as follows:
Outside these eight regions, making up to 34% of the sequence, the identity of the two SEs is 97%, with conserved amino acid substitutions accounting for the remaining differences. Four of these regions are structurally close to the two MHC class II binding sites (B: AA 37-50 (Sequence ID Nos. 11 and 12), D: 71-78 (Sequence ID Nos. 15 and 16), E: 136-149 (Sequence ID Nos. 17 and 18), and G 189-195(Sequence ID Nos. 21 and 22)), and are not likely to interact with the TCR. The additional four regions (A: AA 20-27 (Sequence ID Nos. 9 and 10), C: 60-62 (Sequence ID Nos. 13 and 14), F: 161-176 (Sequence ID Nos. 19 and 20) and H:200-207(Sequence ID Nos. 23 and 24) are located on the edge of the molecule, in the vicinity of the putative TCR binding site, postulated to reside in the groove between the two subdomains. By grafting the individual regions (replacement of amino acid residues that differ), we have now found that the property of SEA-conjugates to induce a cytoxic response as well as potentiating proliferative response in the absence of MHC class II, resides in one region in the TCR binding domain of SEA. This Region (A) is transferable to SEE and has a great impact on activity in the absence of Class II, although limited effects on the Vxcex2 specificity of the superantigen (FIG. 6, Tab.2). All of the regions (A, C, F and H) seem to participate, directly or indirectly, in the interaction with the TCR manifested by an altered stimulatory effect on murine T-cell hybridomas (Tab. 2)
Due to the analogous mode of action it is conceivable that a similar structural separation of these TCRVxcex2 binding properties is at hand also for superantigens analogous to SEA and SEE. The same may also apply within other types of superantigens, in which the binding structures are organised differently. Our discovery has enabled us to outline the construction of chimeric superantigens that potentially are of extremely great value as therapeutic agents.